The impact of fluoxetine and pH values on relaxation of the ternary lipid monolayers

Fluoxetine (FLX), used in the clinic to treat depression, is a well-known cationic amphiphilic antidepressant. To get a deeper insight into the effect of FLX on Langmuir monolayers, in this study the stability and relaxation of 1,2-dioctadecanoyl-sn-glycero-3-phophocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/cholesterol (DSPC/POPC/CHOL) monolayers without and with FLX at different pH values were studied. The experiments involved surface pressure-area (π-A) measurements, mean molecular area-time (A-t) measurements, and atomic force microscope (AFM) analysis. It was found that intermolecular interactions decreased after the addition of FLX in the subphase but increased with increasing pH values. The relaxation of the ternary lipid monolayers with FLX was dominated by dissolution steps, and the dissolution rates decreased with increasing pH values. These findings can be easily confirmed by the analysis of thermodynamic parameters calculated for the investigated films. The data obtained in this study help to understand the effect of drugs on the ternary lipid monolayers from the molecular point of view.     

Polar-group behaviour in mixed monolayers of phospholipids and fusogenic lipids.

1. The surface potentials of mixed monolayers of synthetic phospholipids with lipids that are fusogenic for hen erythrocytes were investigated. 2. At pH 5.6 and 10, but not at pH2, mixed monolayers of the fusogenic lipid, glycerol mono-oleate, with phosphatidylcholine exhibited negative deviations from the ideality rule in surface potential per molecule which were accompanied by negative deviations in mean molecular area. 3. Interactions of this type were not seen with chemically related but non-fusogenic lipids, nor were they found in mixed monolayers of any of the lipids with phosphatidylethanolamine. 4. Experiments with dihexadecyl phosphate and hexadecyltrimethyl-ammonium indicated that the complete head group of phosphatidylcholine is required for its observed behaviour with fusogenic lipids. 5. Bivalent cations (Ca2+, UO2(2+) or Zn2+) in the subphase at pH 5.6 significantly modified the behaviour of mixed monolayers of fusogenic lipids with phospholipids; there was a parallel perturbing effect of fusogenic lipids on interactions between monolayers of phospholipids and bivalent cations. 6. Possible molecular interactions of fusogenic lipids with membrane phospholipids, and the role of Ca2+, are discussed which may be relevant to cell fusion in erythrocytes induced by low-melting lipids in the presence of Ca2+.     

Modulatory effects of different temperatures and Ca2+ concentrations on gangliosides and phospholipids in monolayers at air/water interfaces and their possible functional role

Gangliosides are neuraminic acid-containing glycolipids preferently localized in nervous membranes and showing physicochemical peculiarities, e.g., drastically changing amphiphilic properties by Ca2+ binding. On account of this they are favorite compounds to act as modulators of membraneous organization and functions during synaptic transmission. Lipid monolayers are suitable experimental systems for the study of the surface behavior of amphipatic molecules and therefore are useful to interpret membraneous organization. The surface pressure/area isotherms of monolayers of different individual gangliosides (GM1, GD1a, GD1b, GT1b) of an artificial reconstituted and a natural ganglioside mixture from bovine brain and of ganglioside mixtures from different brain parts of summer- and winter-adapted dsungarian hamsters were compared at three temperatures (11, 20, and 37 degrees C) with egg phosphatidylcholine (PC) and phosphatidylserine (PS) monolayers. The monolayers were formed in a Teflon trough on a triethanolamine/HCl-buffered (pH 7.4) subphase, in some cases containing different amounts of CaCl2. The surface pressure/area isotherms of ganglioside monolayers, in contrast to phospholipids, generally showed slowly rising slopes, with transitions from the liquid-expanded to the liquid-condensed state at a surface pressure of 20-30 mN/m. Ganglioside monolayers, in particular from GD1a or GT1b versus GD1b or from mixtures from summer- versus winter-adapted hamster brain, were differently affected by temperature and/or by Ca2+. PS monolayers were slightly condensed only by Ca2+. PC monolayers, however, were influenced neither by temperature nor by Ca2+. In mixed monolayers of the unpolar natural lipid cholesterol (Ch) and the disialoganglioside GD1a, intermolecular interactions were indicated. Ganglioside monolayers, in contrast to phospholipids, were shown to be easily modulated by temperature and/or Ca2+ ions, thus enabling gangliosides to act as possible membrane modulators, e.g., during synaptic transmission. In particular, the differences concerning the influences of temperature and/or Ca2+ on the surface behavior of ganglioside mixtures from the brain of summer- compared with winter-adapted hamsters are correlated with other physiologically relevant data.     

Packing of ganglioside-phospholipid monolayers: an x-ray diffraction and reflectivity study

Using synchrotron grazing-incidence x-ray diffraction (GIXD) and reflectivity, the in-plane and out-of-plane structure of mixed ganglioside-phospholipid monolayers was investigated at the air-water interface. Mixed monolayers of 0, 5, 10, 20, and 100 mol% ganglioside GM(1) and the phospholipid dipalmitoylphosphatidylethanolamine (DPPE) were studied in the solid phase at 23 degrees C and a surface pressure of 45 mN/m. At these concentrations and conditions the two components do not phase-separate and no evidence for domain formation was observed. X-ray scattering measurements reveal that GM(1) is accommodated within the host DPPE monolayer and does not distort the hexagonal in-plane unit cell or out-of-plane two-dimensional (2-D) packing compared with a pure DPPE monolayer. The oligosaccharide headgroups were found to extend normally from the monolayer surface, and the incorporation of these glycolipids into DPPE monolayers did not affect hydrocarbon tail packing (fluidization or condensation of the hydrocarbon region). This is in contrast to previous investigations of lipopolymer-lipid mixtures, where the packing structure of phospholipid monolayers was greatly altered by the inclusion of lipids bearing hydrophilic polymer groups. Indeed, the lack of packing disruptions by the oligosaccharide groups indicates that protein-GM(1) interactions, including binding, insertion, chain fluidization, and domain formation (lipid rafts), can be studied in 2-D monolayers using scattering techniques.     

Integration of ganglioside GT1b receptor into DPPE and DPPC phospholipid monolayers: an X-ray reflectivity and grazing-incidence diffraction study

Using synchrotron grazing-incidence x-ray diffraction (GIXD) and reflectivity, the in-plane and out-of-plane structures of mixed-ganglioside GT_1b-phospholipid monolayers were investigated at the air-liquid interface and compared with monolayers of the pure components. The receptor GT_1b is involved in the binding of lectins and toxins, including botulinum neurotoxin, to cell membranes. Monolayers composed of 20 mol % ganglioside GT_1b, the phospholipid dipalmitoyl phosphatidylethanolamine (DPPE), and the phospholipid dipalmitoyl phosphatidylcholine (DPPC) were studied in the gel phase at 23°C and at surface pressures of 20 and 40 mN/m, and at pH 7.4 and 5. Under these conditions, the two components did not phase-separate, and no evidence of domain formation was observed. The x-ray scattering measurements revealed that GT_1b was intercalated within the host DPPE/DPPC monolayers, and slightly expanded DPPE but condensed the DPPC matrix. The oligosaccharide headgroups extended normally from the monolayer surfaces into the subphase. This study demonstrated that these monolayers can serve as platforms for investigating toxin membrane binding and penetration.     

Ca2+-induced lateral phase separation in phosphatidic acid/phosphatidylcholine monolayers as revealed by fluorescence microscopy

Phase separation in mixed monolayers of phosphatidylcholine (PC) and pyrene-labeled phosphatidic acid (PA) was observed by fluorescence microscopy on an air/water interface as a function of subphase Ca2+ concentration and lateral packing pressure of the film. Below 45 mN m-1 and in the absence of Ca2+ no indications of phase immiscibility were observed. Addition of 1 mM Ca2+ caused extensive phase separation, which was evident immediately after spreading of the film. Further increase in Ca2+ concentration up to 30 mM increased the pyrene excimer intensity of the separated phosphatidic acid enriched domains. In the presence of Ca2+ (1-30 mM) and at surface pressures below 10 mN m-1 phase separation was always evident. However, as surface pressure exceeded 10 mN m-1, mixing of PC and PA occurred. Upon decompression of the film, phase separation reappeared at surface pressures close to 10 mN m-1. The surface textures of the film before and after the compression and subsequent relaxation were different. Inclusion of 30 mol% cholesterol increased the number and decreased the size of the PA domains. In films containing 50 mol% cholesterol no phase separation could be detected at the resolution available.     

Effect of synthetic surfactants and soapwort (Saponaria officinalis L.) extract on skin-mimetic model lipid monolayers

The effect of a saponin-rich extract from rhizomes of Soapwort (Saponaria officinalis L) and four synthetic surfactants: sodium lauryl sulphate (SLS), sodium laureth sulphate (SLES), ammonium lauryl sulphate (ALS) and cocamidopropyl betaine (CAPB) on two model lipid monolayers is analyzed using surface pressure, surface dilatational rheology and fluorescence microscopy. The following monolayers were employed: dipalmitoylphosphatidylcholine/cholesterol mixture in a molar ratio of 7:3 (DPPC/CHOL) and Ceramide [AP]/stearic acid/cholesterol in a molar ratio of 14:14:10 (CER/SA/CHOL). They mimicked a general bilayer structure and an intercellular lipid mixture, respectively. Both lipid mixtures on Milli-Q water were first compressed to the initial surface pressure, Π₀ = 30 mN/m and then the subphase was exchanged with the respective (bio)surfactant solution at 1% (w/w). All four synthetic surfactants behaved in a similar way: they increased surface pressure to about 40 mN/m and reduced the storage modulus of surface dilational surface rheology, E′, to the values close to zero. The corresponding fluorescence microscopy pictures confirmed that the lipids mimicking the stratum corneum components were almost completely removed by the synthetic surfactants under the present experimental conditions. The components of the Soapwort extract (SAP) increased surface pressure to significantly higher values than the synthetic surfactants, but even more spectacular increase was observed for the storage modulus of the SAP-penetrated lipid monolayers (up to E′= 715 mN/m).     

Domain structure and molecular conformation in annexin V/1,2-dimyristoyl-sn-glycero-3-phosphate/Ca2+ aqueous monolayers: a Brewster angle microscopy/infrared reflection-absorption spectroscopy study.

Annexins comprise a family of proteins that exhibit a Ca2+-dependent binding to phospholipid membranes that is possibly relevant to their in vivo function. Although substantial structural information about the ternary (protein/lipid/Ca2+) interaction in bulk phases has been derived from a variety of techniques, little is known about the temporal and spatial organization of ternary monolayer films. The effect of Ca2+ on the interactions between annexin V (AxV) and anionic DMPA monolayers was therefore investigated using three complementary approaches: surface pressure measurements, infrared reflection-absorption spectroscopy (IRRAS), and Brewster angle microscopy (BAM). In the absence of Ca2+, the injection of AxV into an aqueous subphase beneath a DMPA monolayer initially in a liquid expanded phase produced BAM images revealing domains of protein presumably surrounded by liquid-expanded lipid. The protein-rich areas expanded with time, resulting in reduction of the area available to the DMPA and, eventually, in the formation of condensed lipid domains in spatial regions separate from the protein film. There was thus no evidence for a specific binary AxV/lipid interaction. In contrast, injection of AxV/Ca2+ at a total Ca2+ concentration of 10 microM beneath a DMPA monolayer revealed no pure protein domains, but rather the slow formation of pinhead structures. This was followed by slow (>2 h) rigidification of the whole film accompanied by an increase in surface pressure, and connection of solid domains to form a structure resembling strings of pearls. These changes were characteristic of this specific ternary interaction. Acyl chain conformational order of the DMPA, as measured by nu(sym)CH2 near 2850 cm(-1), was increased in both the AxV/DMPA and AxV/DMPA/Ca2+ monolayers compared to either DMPA monolayers alone or in the presence of Ca2+. The utility of the combined structural and temporal information derived from these three complementary techniques for the study of monolayers in situ at the air/water interface is evident from this work.     

Influence of calcium, cholesterol, and unsaturation on lecithin monolayers

Surface pressures and potentials of mixed monolayers of dicetyl phosphate-cholesterol, dipalmitoyl lecithin-cholesterol, egg lecithin-cholesterol, and phosphatidic acid-cholesterol were measured. The surface potential is shown to be a more reliable parameter for the study of interactions in monolayers than the surface pressure. Monolayers of dicetyl phosphate-cholesterol follow the additivity rule for area/molecule whereas lecithin-cholesterol monolayers deviate from it. The reverse is true for the additivity rule with regard to surface potential/molecule. Thus, the surface potential indicates that there is no interaction (or complex formation) between lecithin and cholesterol, but that there is ion-dipole interaction between dicetyl phosphate and cholesterol, as well as between phosphatidic acid and cholesterol. The apparent condensation of mixed monolayers of lecithin when cholesterol is added is explained by a consideration of molecular cavities or vacancies caused by thermal motion of the fatty acyl chains, the size of these cavities being influenced by the length and degree of saturation (especially the proportion of monounsaturation) of the fatty acyl chains and the extent of compression of the monolayer. The cholesterol molecules occupy these cavities and therefore cause no proportional increase in area/molecule in the mixed monolayers. Monolayers are liquefied by the presence of cholesterol as well as of unsaturated fatty acyl chains; in contrast, Ca(++)tends to solidify lecithin monolayers. The available evidence suggests that cholesterol can both impart fluidity to the monolayer and occupy the molecular cavities caused by the fatty acyl chains.     

Pulmonary surfactant protein A interacts with gel-like regions in monolayers of pulmonary surfactant lipid extract

Epifluorescence microscopy was used to investigate the interaction of pulmonary surfactant protein A (SP-A) with spread monolayers of porcine surfactant lipid extract (PSLE) containing 1 mol % fluorescent probe (NBD-PC) spread on a saline subphase (145 mM NaCl, 5 mM Tris-HCl, pH 6.9) containing 0, 0.13, or 0.16 microg/ml SP-A and 0, 1.64, or 5 mM CaCl(2). In the absence of SP-A, no differences were noted in PSLE monolayers in the absence or presence of Ca(2+). Circular probe-excluded (dark) domains were observed against a fluorescent background at low surface pressures (pi approximately 5 mN/m) and the domains grew in size with increasing pi. Above 25 mN/m, the domain size decreased with increasing pi. The amount of observable dark phase was maximal at 18% of the total film area at pi approximately 25 mN/m, then decreased to approximately 3% at pi approximately 40 mN/m. The addition of 0.16 microg/ml SP-A with 0 or 1.64 mM Ca(2+) in the subphase caused an aggregation of dark domains into a loose network, and the total amount of dark phase was increased to approximately 25% between pi of 10-28 mN/m. Monolayer features in the presence of 5 mM Ca(2+) and SP-A were not substantially different from those spread in the absence of SP-A, likely due to a self-association and aggregation of SP-A in the presence of higher concentrations of Ca(2+). PSLE films were spread on a subphase containing 0.16 microg/ml SP-A with covalently bound Texas Red (TR-SP-A). In the absence of Ca(2+), TR-SP-A associated with the reorganized dark phase (as seen with the lipid probe). The presence of 5 mM Ca(2+) resulted in an appearance of TR-SP-A in the fluid phase and of aggregates at the fluid/gel phase boundaries of the monolayers. This study suggests that SP-A associates with PSLE monolayers, particularly with condensed or solid phase lipid, and results in some reorganization of rigid phase lipid in surfactant monolayers.     

Large-scale recrystallization of the S-layer of Bacillus coagulans E38-66 at the air/water interface and on lipid films.

S-layer protein isolated from Bacillus coagulans E38-66 could be recrystallized into large-scale coherent monolayers at an air/water interface and on phospholipid films spread on a Langmuir-Blodgett trough. Because of the asymmetry in the physiochemical surface properties of the S-layer protein, the subunits were associated with their more hydrophobic outer face with the air/water interface and oriented with their negatively charged inner face to the zwitterionic head groups of the dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine (DPPE) monolayer films. The dynamic crystal growth at both types of interfaces was first initiated at several distant nucleation points. The individual monocrystalline areas grew isotropically in all directions until the front edge of neighboring crystals was met. The recrystallized S-layer protein and the S-layer-DPPE layer could be chemically cross-linked from the subphase with glutaraldehyde.     

Differential effects of surfactant protein A on regional organization of phospholipid monolayers containing surfactant protein B or C

Epifluorescence microscopy combined with a surface balance was used to study monolayers of dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylglycerol (PG) (8:2, mol/mol) plus 17 wt % SP-B or SP-C spread on subphases containing SP-A in the presence or absence of 5 mM Ca(2+). Independently of the presence of Ca(2+) in the subphase, SP-A at a bulk concentration of 0.68 microg/ml adsorbed into the spread monolayers and caused an increase in the molecular areas in the films. Films of DPPC/PG formed on SP-A solutions showed a pressure-dependent coexistence of liquid-condensed (LC) and liquid-expanded (LE) phases. Apart from these surface phases, a probe-excluding phase, likely enriched in SP-A, was seen in the films between 7 mN/m < or = pi < or = 20 mN/m. In monolayers of SP-B/(DPPC/PG) spread on SP-A, regardless of the presence of calcium ions, large clusters of a probe-excluding phase, different from probe-excluding lipid LC phase, appeared and segregated from the LE phase at near-zero surface pressures and coexisted with the conventional LE and LC phases up to approximately 35 mN/m. Varying the levels of either SP-A or SP-B in films of SP-B/SP-A/(DPPC/PG) revealed that the formation of the probe-excluding clusters distinctive for the quaternary films was influenced by the two proteins. Concanavalin A in the subphase could not replace SP-A in its ability to modulate the textures of films of SP-B/(DPPC/PG). In films of SP-C/SP-A/(DPPC/PG), in the absence of calcium, regions consisting of a probe-excluding phase, likely enriched in SP-A, were detected at surface pressures between 2 mN/m and 20 mN/m in addition to the lipid LE and LC phases. Ca(2+) in the subphase appeared to disperse this phase into tiny probe-excluding particles, likely comprising Ca(2+)-aggregated SP-A. Despite their strikingly different morphologies, the films of DPPC/PG that contained combinations of SP-B/SP-A or SP-C/SP-A displayed similar distributions of LC and LE phases with LC regions occupying a maximum of 20% of the total monolayer area. Combining SP-A and SP-B reorganized the morphology of monolayers composed of DPPC and PG in a Ca(2+)-independent manner that led to the formation of a separate potentially protein-rich phase in the films.     

Interactions in mixed monolayers between distearoyl-l-phosphatidylethanolamine, rod outer segment phosphatidylethanolamine and all-trans retinal. Effect of pH

The interactions in mixed monolayers between distearoyl-l-phosphatidylethanolamine, natural phosphatidylethanolamine purified from bovine rod outer segments and all-trans retinal have been studied at the nitrogen/water interface at 21.0 ± 0.5°C. Seven mixtures of each phospholipid with all-trans retinal, covering the whole range of molar fractions, were studied. The monolayers were spread on a 1·10⁻³ M phosphate buffer subphase at three different pH values, 5.5, 7.1 and 8.2. The results for the two series of mixtures are strikingly different. The surface phase rule shows that all-trans retinal is miscible with the natural phospholipid at the interface. Small, negative deviations with respect to the additivity rule are observed in this case. The excess free energies of mixing were also calculated as a function of concentration for this system at four different surface pressures, 5, 7, 10 and 13 mN·m⁻¹. They are negative for the four surface pressures considered and symmetrical with respect to the mole fraction. On the other hand, when distearoyl-l-phosphatidylethanolamine is mixed with all-trans retinal, the components are no longer miscible at the interface. This marked difference in behaviour between the two lipids reflects the importance of hydrophobic interactions in the mixed monolayers of phospholipids with retinals. Furthermore, for the two series of mixtures, the surface pressure isotherms do not show any significant shift when the subphase pH is changed from 5.5 to 8.2. This behaviour raises questions about the formation of a Schiff base between phosphatidylethanolamine and retinal at the interface. It is suggested that, owing to the nature of the disk membranes, such an effect would also be observed in vivo. The possible implications of this are discussed, particularly with respect to questions pertaining to the stability of the retinal chromophore.     

Adsorption of apolipoprotein A-IV to phospholipid monolayers spread at the air/water interface. A model for its labile binding to high density lipoproteins.

The mechanisms that mediate the labile binding of apolipoprotein A-IV (apoA-IV) to high density lipoproteins (HDL) are not known. We therefore used a surface balance and surface radioactivity detector to investigate the adsorption of apoA-IV to egg phosphatidylcholine monolayers spread at the air/water interface. ApoA-IV bound rapidly and reversibly to phospholipid monolayers and generated a maximum increase in surface pressure of 19 millinewtons (mN)/m at a subphase concentration of 2 x 10(-5) g/dl. Binding decreased linearly with increasing initial surface pressure; at pressures greater than 28-29 mN/m, apoA-IV could no longer penetrate the lipid monolayer. The area occupied by the amino acid residues in apoA-IV reached an unusually low limiting molecular area of 10-12 A2/residue at surface saturation. The surface pressure of native HDL3 was calculated to be 33 mN/m, and it rapidly decreased with the action of lecithin:cholesterol acyltransferase on the particle surface. We conclude that the surface activity of apoA-IV is lower than that of any other human apolipoprotein; its binding and surface conformation are particularly sensitive to pressure; and at saturation, a significant portion of the molecule is excluded from the interface. The exclusion pressure of apoA-IV may be only slightly lower than the surface pressure of HDL; in vivo, the action of lecithin:cholesterol acyltransferase and lipid transfer proteins may cause the HDL3 surface pressure to oscillate about a narrow range that spans the exclusion pressure of apoA-IV. The resultant labile association of apoA-IV and HDL may be of central importance to its role in lipoprotein metabolism.     

Chitosan as a lipid binder: a langmuir monolayer study of chitosan-lipid interactions

Owing to its distinct chemico-biological properties, chitosan, a cationic biopolymer, offers a great potential in multifarious bioapplications. One such application is as a dietary antilipidemic supplement to be used to reduce obesity/overweight and to lower cholesterol. The lipid-binding efficiency of chitosan, however, remains debatable. Accordingly, in this study we investigated the interactions of chitosan with selected lipids, cholesterol and fatty acids, the latter including saturated (stearic acid) and unsaturated (oleic, linoleic, alpha-linolenic) acids. The experiments were performed with the Langmuir monolayer technique, in which surface pressure-area isotherms were recorded for the lipid monolayers spread on the acetate buffer pH 4.0 subphase in the absence and presence of chitosan. We found that the presence of chitosan in the subphase strongly influenced the shape and location of the isotherms, proving that there existed attractions between chitosan and lipid molecules. The attractions were revealed by changes of the molecular organization of the monolayers. The common feature of these changes was that all the monolayers studied underwent expansion, in each case reaching saturation with increasing chitosan concentration. In agreement with the lipid molecular structures, the highest expansions were observed for the most unsaturated fatty acids, linoleic and alpha-linolenic, the lowest for stearic acid, with oleic acid and cholesterol being the intermediate cases. By contrast, the main distinguishing feature of these changes was that, although none of the monolayers studied changed its state when completely saturated with chitosan, compared to the parent ones the compactness of the monolayers was modified. The solid monolayers of stearic acid and cholesterol were loosened, whereas those of all the unsaturated acids, liquid in nature, were tightened. On the basis of these results we tentatively propose a mechanism of the chitosan action that includes both electrostatic and hydrophobic lipid-chitosan interactions as well as hydrogen bonding between them.     

The interaction of cytochrome c with monolayers of phosphatidylethanolamine

1. The interaction between [(14)C]carboxymethylated cytochrome c and monolayers of egg phosphatidylethanolamine at the air/water interface has been investigated by measurements of surface radioactivity, pressure and potential. 2. On adding (14)C-labelled cytochrome c to the subphase under monolayers with a surface pressure below 24dynes/cm. there was an initial surface pressure increment as the protein penetrated, followed by an adsorption that could be detected only by a continued increase in the surface radioactivity. 3. Above film pressures of 24dynes/cm. only adsorption was observed, i.e. an increment in surface radioactivity with none in surface pressure. 4. The changes in surface parameters with penetration of cytochrome c added to the subphase were indirectly proportional to the initial pressure of the monolayer. With hydrogenated phosphatidylethanolamine the constant of proportionality was increased but penetration again ceased at 24dynes/cm. 5. On compressing a phosphatidylethanolamine film containing penetrated cytochrome c to 40dynes/cm. only a proportion of the protein was ejected on a subphase of 10mm-sodium chloride, whereas on a subphase of m-sodium chloride nearly all the protein was lost. 6. With both penetration and adsorption only a small proportion of the added cytochrome c interacted with the phospholipid films, and initially the amount bound was proportional to the added protein concentration. There was no evidence of a stoicheiometric relationship between the protein and phospholipid or the build-up of multilayers. The bonded protein was not released by removing cytochrome c from the subphase. 7. The addition of m-sodium chloride to the subphase delays the rate of protein penetration into low-pressure films, but the final surface-pressure increment is not appreciably decreased. In contrast, m-sodium chloride almost completely stops adsorption on to films at all pressures. 8. When sodium chloride is added to the subphase below cytochrome c adsorbed to monolayers at high pressures, so that the final concentration is 1m, only a proportion of the protein is desorbed and this decreases as the time of the interaction increases. This indicates that adsorption is initially electrostatic, followed by the formation of non-ionic bonds. 9. Alteration of the subphase pH under a high-pressure film leads to a steady increase in adsorption from pH3 to 8.5 followed by a rapid fall to zero adsorption at pH11. 10. The penetration into phospholipid monolayers at 10dynes/cm. shows a rate that is consistent with the relative electrostatic status of the two components of the interaction as the subphase pH is varied between 3 and 10.5. The final equilibrium penetration shows a pronounced peak in the increments of surface pressure at pH9.0 although a similar peak is not observed in the surface radioactivity. This indicates that more residues of the protein are penetrating into the film at about this pH. 11. Determinations were made of the electrophoretic mobilities of phosphatidylethanolamine particles both alone and after interaction with cytochrome c. 12. The electrophoretic mobilities of cytochrome c adsorbed on lipid particles showed an isoelectric point below that of cytochrome c. This and the observations on the monolayers suggest that, with cytochrome c, protein-protein interactions are weak compared with other proteins.     

Conformational changes of the lecithin headgroup in monolayers at the air/water interface

The headgroup conformation of the phospholipid dipalmitoyl-glycero-phosphocholine (DPPC) in monolayers at the air/water interface has been studied by neutron reflection in the fluid like liquid-expanded (LE) and in the crystal like solid (S) phase. Information on the headgroup conformation in the two phases has been obtained by scattering contrast variation of the lipid monolayer using four differently deuterated species of DPPC: perdeuterated, chain perdeuterated, choline group perdeuterated and selectively headgroup deuterated. Since the measurements were done mainly on a subphase of null reflecting water (i.e. water scattering contrast matched to the air) there is no subphase contribution to reflectivity and the simplest one layer model can be employed for the data analysis, thus minimising the number of free parameters. A remarkable change of the headgroup orientation was observed between the LE and the S phase. We found that the phosphate-nitrogen dipole of the DPPC headgroup exhibits an in-plane orientation with respect to the monolayer in the LE phase but it assumes a more parallel orientation to the surface normal at lateral pressures above 30 mN/m (S phase). Moreover, this conformational change is accompanied by a significant alteration of the headgroup hydration.Abbreviations DPPC Dipalmitoyl-Phosphatidylcholine - DMPC Dimyristoyl-Phosphatidylcholine - DPPE Dipalmitoyl-Phosphatidylethanolamine - DMPE Dimyristoyl-Phosphatidylethanolamine - DMPA Dimyristoyl-Phosphatic Acid - DMPG Dimyristoyl-Phosphatidylglycerol Correspondence to: T M. Bayed     

The role of surface charge in the activation of the classical and alternative pathways of complement by liposomes

We have studied the complement-activating properties of liposomes. We show that surface charge is a key determinant of complement-activating liposomes. The nature of the charge, whether negative or positive, appears to dictate which pathway of the complement system is activated. Phosphatidylcholine:cholesterol (PC:CHOL, 55:45 mol/mol) liposomes were made to exhibit a positive or negative surface charge by the addition of cationic or anionic lipids, respectively. Normal human or guinea pig serum was incubated with liposomes, followed by determining the residual hemolytic activity of the serum as a measure of complement activation. Negatively charged liposomes containing phosphatidyl-glycerol, phosphatidic acid, cardiolipin, phosphatidylinositol, or phosphatidylserine activated complement in a Ca(2+)-dependent manner suggesting activation occurred via the classical pathway. Positively charged liposomes containing stearylamine or 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane activated complement via the alternative pathway. Neutral liposomes, PC:CHOL (55:45) and PC:CHOL:dipalmitoylphosphatidylethanolamine (35:45:20), failed to activate complement as measured by the hemolytic assays. We show that unsaturated liposomes are more potent complement activators than saturated liposomes and that 45 mol% cholesterol promotes complement protein-liposome interactions. Immunoblot analysis of phosphatidylglycerol-containing liposomes showed that C3b and C9 were associated with these liposomes. Thus, the complement consumption measured in the hemolytic assays represents active cleavage of the complement components and not passive adsorption to the liposome surface. These studies suggest that membranes composed of net charged phospholipids can activate the complement system. This observation underlines the importance in biologic membranes of complement regulatory proteins that protect normal cells from complement attack.     

Probing the modes of antibacterial activity of chitosan. Effects of pH and molecular weight on chitosan interactions with membrane lipids in Langmuir films

Chitosan, a cationic biopolymer derived from chitin, has been described as having antibacterial activity. The modes of this activity, however, have not been established. One mode proposed is that chitosan perturbs bacterial cell membranes. To validate this proposal, in this study we investigated chitosan interactions with lipids in Langmuir monolayers as model membranes. The interactions were assessed by monitoring differences in the shape of the compression isotherms measured in the absence and presence of chitosan in the subphase (acetate buffer). To appraise the contribution of electrostatic interactions versus hydrogen bonding and hydrophobic interactions, three membrane lipids differing in charge were studied-anionic dipalmitoylphosphatidylglycerol (DPPG), zwitterionic dipalmitoylphosphatidylcholine (DPPC), and neutral cholesterol-and the pH of the subphase was varied between 3.5 and 6.0. In addition, the impact of the molecular weight of chitosan on the interactions was assessed at pH 3.5. It was found that while chitosan had a negligible effect on DPPC monolayers over the pH range studied, it distinctly affected DPPG and cholesterol monolayers. The effect on DPPG was found to decrease with increasing pH, that at pH 3.5 being ascribed to the charge-mediating action of chitosan on the local ionic environment and that at higher pHs to the intercalation of chitosan to the monolayers. Practically independent of pH, the effect of chitosan on cholesterol was accounted for by the formation of cholesterol-chitosan hydrogen bonds. Chitosan of lower molecular weight facilitated the interactions with all the three lipids studied. The results obtained may be helpful in identifying the mode of antibacterial activity of chitosan versus other modes that involve the disturbance of cell life cycles.     

Incorporation of dehydrodieugenol,a neolignan isolated from Nectandra leucantha (Lauraceae), in lipid Langmuir monolayers as biomembrane models

Dehydrodieugenol, a neolignan isolated from the Brazilian plant Nectandra leucantha (Lauraceae) with reported antiprotozoal and anticancer activity, was incorporated in Langmuir monolayers of selected lipids as cell membrane models, aiming to comprehend its action mechanism at the molecular level. The interaction of this compound with the lipids dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylserine (DPPS), and dipalmitoylphosphatidylglycerol (DPPG) was inferred through tensiometry, infrared spectroscopy, and Brewster angle microscopy. The interactions had different effects depending on the chemical nature of the lipid polar head, with expansion for DPPC monolayers, condensation for DPPE, and expansion (at low surface pressures) followed by the overlap of the isotherms (at high surface pressure values) for DPPS and DPPG. Effects caused by dehydrodieugenol in the negatively charged lipids were distinctive, which was also reflected in the hysteresis assays, surface potential-area isotherms, and rheological measurements. Infrared spectroscopy indicated that the drug interaction with the monolayer affects not only the polar groups, but also the acyl lipid chains for all lipids. These results pointed to the fact that the interaction of the drug with lipid monolayers at the air-water interface is modulated by the lipid composition, mainly considering the polar head of the lipids, as well as the hydrophobicity of the lipids and the drug. As negatively charged lipids pointed to distinctive interaction, we believe this can be related to the antiprotozoal and anticancer properties of the compound.